Recent large-scale genomic analyses have revealed highly recurrent somatic single nucleotide variants (sSNVs) occurring within the majority of the common cancer subtypes. Incorporating molecular-based classifications with conventional tissue-based pathologic diagnosis will continue to guide decision-making for treatment.
For example, in glioma the recurrent somatic R132H mutation in Isocitrate Dehydrogenase 1 (IDH1) is detected in 75% of grade II and III astrocytomas and two separate mutations in the promoter region of Telomerase Reverse Transcriptase (TERT) are detected in 83% of primary glioblastoma (GBM, grade IV astrocytoma) (P. J. Killela et al., Proc. Natl. Acad. Sci. U.S.A. 110, 6021-6026 (2013), C. Koelsche et al., Acta Neuropathol. (Berl.). 126, 907-915 (2013), N. Nonoguchi et al., Acta Neuropathol. (Berl.). 126, 931-937 (2013). Oligodendrogliomas are highly chemotherapy-sensitive gliomas characterized by loss of heterozygosity of chromosome 1p and 19q, but recent studies have found that 80% of these are also defined by concurrent mutations in IDH1 and TERT promoter (P. J. Killela et al., Proc. Natl. Acad. Sci. U.S.A. 110, 6021-6026 (2013), N. Sabha et al., Neuro-Oncol. (2014), doi:10.1093/neuonc/not299., H. Arita et al., Acta Neuropathol. (Berl.). 126, 267-276 (2013)). The classification of glioma by these recurrent mutations in IDH1 and TERT promoter closely mirrors conventional histopathology, but serves as a stronger predictor of response to adjuvant therapy and overall survival than conventional pathology (P. J. Killela et al., Oncotarget (2014), A. K.-Y. Chan et al., Mod. Pathol. Off J. U.S. Can. Acad. Pathol. Inc (2014), doi:10.1038/modpathol.2014.94). This latter point is critical for appropriate clinical trial design and patient stratification.
World Health Organization (WHO) grade II diffuse gliomas are a group of slow-growing primary central nervous system tumors, including oligodendrogliomas, astrocytomas and oligoastrocytomas. Grade II diffuse gliomas are clinically indolent lesions for which progression free and overall patient survival positively correlates with maximal upfront resection (Smith J S, et al., J Clin Oncol Off J Am Soc Clin Oncol 2008; 26(8):1338-45; Yordanova Y N, et al., J Neurosurg 2011; 115(2):232-9; Jakola A S, et al., JAMA 2012; 308(18):1881-8; Potts M B, et al., J Neurosurg 2012; 116(2):365-72; Beiko J, et al., Neuro-Oncol 2014; 16(1):81-91). The initial surgical procedure can therefore be both diagnostic and therapeutic, but accurate intraoperative diagnosis is essential in determining whether to continue with more aggressive surgical resection. However, as a consequence of the tumor location, infiltrative growth, low cellularity and often small stereotactic biopsy specimens, frozen intraoperative pathological assessment of low-cellularity diffuse gliomas can be challenging (Glantz M J, et al., Neurology 1991; 41(11):1741-4; Jackson R J, Fuller G N, Abi-Said D, et al.; 3(3):193-200; Regragui A, et al., Neurochirurgie 2003; 49(2-3 Pt 1): 67-72). This diagnostic dilemma often requires clarification by final pathologic analysis several days after the diagnostic biopsy, including immunohistochemistry (IHC) for Ki67, TP53, IDH1 R132H or even targeted sequencing. Once the diagnosis is firmly established, patients may then require a second neurosurgical procedure for definitive resection.
Recent studies have demonstrated that diffuse gliomas harbor characteristic recurrent somatic mutations in IDH1 (present in >80% of diffuse astrocytomas, oligodendrogliomas, oligoastrocytomas and secondary glioblastoma (GBM); Yan H, et al., N Engl J Med 2009; 360(8):765-73) and the TERT promoter (similarly present in up to 80% of oligodendrogliomas and primary GBM; Arita H, et al., Acta Neuropathol (Berl) 2013; 126(2):267-76; Killela P J, et al., Proc Natl Acad Sci USA 2013; 110(15):6021-6; Nonoguchi N, et al., Acta Neuropathol (Berl) 2013; 126(6):931-7). An intraoperative assay that could rapidly and accurately identify these recurrent molecular features could augment “frozen section” histopathologic analysis, improve intraoperative diagnosis and accelerate real-time decision-making in the management of grade II diffuse gliomas.
Melanoma is a highly prevalent neoplasm that is also characterized by recurrent missense sSNVs leading to activating mutations in either B-Raf or NRAS (E. Hodis et al., Cell. 150, 251-263 (2012)). Benign nevi have also been found to harbor BRAF V600E, which reduces the specificity of the alteration for distinguishing these nevi from malignant pathologies (J. M. Taube, S. Begum, C. Shi, J. R. Eshleman, W. H. Westra, Am. J. Surg. Pathol. 33, 568-571 (2009)). However, recent whole genome analyses have revealed that primary melanoma and basal cell cancer are uniquely characterized by recurrent mutations in the promoter region of TERT (F. W. Huang et al., Science. 339, 957-959 (2013), S. Horn et al., Science. 339, 959-961 (2013), J. Vinagre et al., Nat. Commun. 4, 2185 (2013)), and, thus, more specifically discriminates true neoplastic pathology from benign lesions.
Somatic single nucleotide variants targeting the Q61 codon of NRAS occurs in 10-25% of cutaneous melanoma (Tsao, H., et al. (2012); Genes Dev., 26:1131-1155). Patients undergoing therapies targeting mutant BRAF have been found to develop resistance through mechanisms that result in mutations to NRAS Q61 codon (Van Allen E, et al. (2014); Can Disc., 4:94). Furthermore, patients with NRAS-mutant melanoma have a better response to immunotherapy than those with mutant BRAF (Johnson, D B, et al. (2015); Cancer Immunol Res., 3(3):288-295) therefore monitoring the blood for evidence of these alterations can have great prognostic value.
Diffuse intrinsic pontine gliomas (DIPG) are diffusely infiltrative malignant glial neoplasms that arise in the brainstem during childhood (Panditharatna E., et al. (2015); Cancer Genet. 208, 367-373). These tumors are highly aggressive and ultimately fatal. Though there is a growing consensus toward obtaining biopsies of patients with suspected DIPG (Walker, D. A. et. al. (2013); Neuro-oncology 4, 462-468), concerns remain regarding the risks of routine brainstem biopsies in children. A large percentage of pediatric high-grade gliomas have recently been found to harbor recurrent mutations in H3F3A, including more than 70% of diffuse intrinsic pontine glioma, one-third of pediatric glioblastoma and nearly 20% of pediatric anaplastic astrocytoma (Schwartzentruber, J. et al. (2012); Nature 482, 226-231, Wu, G. et al. (2012); Nat. Genet. 44, 251-253). Improving the diagnostic success rate for diffuse gliomas while decreasing the risks associated with biopsy of critical brain structures, as in cases of suspected DIPG, could significantly improve the management of diffuse gliomas in adults and children. The detection of genomic alterations that define diffuse gliomas, including DIPGs, from small volumes of Cerebrospinal Fluid (CSF), would allow for minimally invasive diagnosis and monitoring of response to cancer-directed therapies using CSF specimens obtained by lumbar punctures.
Uveal melanomas are malignancies originating within the melanocytes of the eye involving the iris, ciliary body or choroid. Patients with uveal melanoma have an approximately 50% likelihood of developing metastases and of those, the median survival rate is 2 to 15 months (Augsburger J., et al. (2009); Am J. Opthalmol, 148:119-127, and van den Bosch, T., et al. (2010); Dermatol Res Pract, 2010: 360136). In upwards of 80% of uveal melanomas, somatic single nucleotide variants in GNAQ or GNA11 are found to occur and have been shown to lead to the activation of the MAPK pathway (Chen X., et al., (2014); Oncogene, 33:4724-4734, Van Raamsdonk C D, et al (2010); N Engl J Med, 363:2191-2199, Van Raamsdonk C D, et al. (2009); Nature 457:599-602). Inhibition of the MAPK pathway shows promise in leading to improved progression free survival (Carvajal R D, et al. (2014); Jama 311:2397-2405, however predicting response is hindered by the location of the tumor. Improved diagnostic success would be achieved through monitoring the blood of patients with uveal melanoma by the detection of alterations to GNAQ and GNA11.
Timely and accurate diagnosis of cancer subtypes is critical for appropriately stratifying patients for real-time intraoperative decision-making. For instance, a “frozen section” during neurosurgical resection of malignant gliomas can facilitate intraoperative decision-making, however, the concordance between frozen and permanent histopathology has been reported to be ˜90% (A. Regragui, et al., Neurochirurgie. 49, 67-72 (2003)) due to secondary to small or unrepresentative sample size, low tumor purity and disruption of histologic architecture (M. J. Glantz et al., Neurology. 41, 1741-1744 (1991), R. J. Jackson et al., Neuro-Oncol. 3, 193-200 (2001), T. P. Plesec, R. A. Prayson, Arch. Pathol. Lab. Med. 131, 1532-1540 (2007), B. Y. S. Kim et al., J. Neurooncol. (2014), doi:10.1007/s11060-014-1451-0). Molecular based cancer diagnostic assays are also important for clustering patients of similar prognoses for clinical trial design (I. K. Mellinghoff et al., N. Engl. J. Med. 353, 2012-2024 (2005)) and can guide targeted therapies in the management of malignant neoplasms.
There is a need for translation of this genomic knowledge to provide sensitive and rapid molecular-based pathologic determinations in a timeframe that can impact surgical decision-making and to facilitate non-invasive monitoring of disease progression and treatment response.